This invention relates to in vitro culturing processes for regenerating tissue, e.g. shoots, somatic embryos or roots, of tree genotypes at high frequency, to the use of such processes for producing tree genotypes which tolerate normally lethal dosages of toxic chemicals, such as herbicides, and to the tolerant tree genotypes thus produced.
Trees are being increasingly exposed to chemical stress because of urbanization, industrialization, and intensive forest and horticultural management practices. Chemical stress is produced by such chemicals as sulfur dioxide, ozone, nitrogen oxides, mineral salts, heavy metals, and acid precipitation. Chemical stress can damage or kill trees.
Herbicides are another cause of chemical stress to trees. Yet, the use of herbicides is desirable in tree cultivation for preparing planting sites, reducing weed competition, converting sites in order to plant other species, and additional purposes. As tree cultivation intensifies, herbicide use is increasing in order to make production more economical. Cultivation with herbicides is less expensive than mechanical methods which may not even work.
Herbicides could be used more and their use would be mere efficient if the tree crops exposed thereto are tolerant of the herbicides. This is particularly so in the case of the genus Populus, a genus of great value to the forest industry, trees of this genus being the object of much use in short rotation intensive culture to yield pulp, fiberboard and fuelwood. Unfortunately, Populus trees are sensitive to many herbicides. Naturally occurring Populus trees do not have commercially useful levels of tolerance to such environmentally safe, broad-spectrum herbicides as glyphosate, sulfonylureas (sulfometuron methyl), or imidazolinones. In the absence of herbicides, weed competition limits the plantation establishment of Populus trees even though the weeds can be killed by such herbicides. Present Populus weed control methods involve labor-intensive shielding methods or are limited to use during cessation of the growth of the Populus crop.
Chemical stress tolerance and other traits are difficult or impossible to obtain in trees by traditional crossbreeding. If higher plant genes for such traits exist, they occur at such low frequencies in tree populations that they cannot be economically selected, if at all. As a result, it is difficult or impossible to obtain tolerant parents for breeding. In addition, trees take a long time to reach sexual maturity, usually many years. Thus, traditional breeding for a trait, such as chemical stress tolerance, would take years, perhaps a century. Additionally, often nothing is known about the modes of inheritance of a trait in trees. This lack of knowledge further complicates breeding.
In vitro tissue culturing offers a practical alternative for developing genetically modified individuals of tree species. Variants can be produced during in vitro culture propagation whereby all the progeny of a given plant material are not identical. This variation resulting solely from the in vitro culturing process is known as somaclonal variation. Somaclonal variation may be Mendelian (quantitative) and non-Mendelian (qualitative), and may arise from preexisting cellular genetic variation or variation arising in the culture process, or both. Somaclonal variants may be expressed, described, and tested from a single passage in microculture and may have desirable economic traits that can be exploited directly through their sexually or vegetatively propagated progenies, or indirectly by further somaclonal or traditional breeding programs.